Concentration-dependent oligomerization of cross-linked complexes between ferredoxin and ferredoxin–NADP⁺ reductase

Highlights

• Cross-linked complexes of ferredoxin (Fd) and Fd–NADP⁺ reductase form oligomers.

• In the crystal structures, Fd- and FNR moieties swap across the molecules.

• The complexes exhibit concentration-dependent oligomerization at sub-milimolar order.

Abstract

Ferredoxin–NADP⁺ reductase (FNR) forms a 1:1 complex with ferredoxin (Fd), and catalyzes the electron transfer between Fd and NADP⁺. In our previous study, we prepared a series of site-specifically cross-linked complexes of Fd and FNR, which showed diverse electron transfer properties. Here, we show that X-ray crystal structures of the two different Fd–FNR cross-linked complexes form oligomers by swapping Fd and FNR moieties across the molecules; one complex is a dimer from, and the other is a successive multimeric form. In order to verify whether these oligomeric structures are formed only in crystal, we investigated the possibility of the oligomerization of these complexes in solution. The mean values of the particle size of these cross-linked complexes were shown to increase with the rise of protein concentration at sub-milimolar order, whereas the size of dissociable wild-type Fd:FNR complex was unchanged as analyzed by dynamic light scattering measurement. The oligomerization products were detected by SDS–PAGE after chemical cross-linking of these complexes at the sub-milimolar concentrations. The extent and concentration-dependent profile of the oligomerizaion were differentiated between the two cross-linked complexes. These results show that these Fd–FNR cross-linked complexes exhibit concentration-dependent oligomerization, possibly through swapping of Fd and FNR moieties also in solution. These findings lead to the possibility that some native multi-domain proteins may present similar phenomenon in vivo.

Introduction

Evidences have been shown that some proteins coexist in more than one oligomeric state under physiological conditions, and the ratio of these alternate oligomers can be varied depending on the protein composition or environment [1], [2], [3], [4]. One of the common mechanisms for protein oligomerization is domain swapping, which works by converting an intramolecular interface in the monomer to an intermolecular interface between subunits in the oligomer [4]. More than 100 domain-swapped structures have been characterized [5], but the functional and physiological relevance as well as the mechanism of the swapping is often not understood. However, growing evidence supports the hypothesis that domain swapping has diverse biological functions in oligomerization [6], [7], [8], [9], [10], [11]; some potential advantages include higher local concentrations of active sites, larger binding surfaces, new active sites at subunit interfaces, the possibility of allosteric control, and economic ways to produce large protein interaction networks and molecular machineries [8], [12].

Ferredoxin (Fd) and Fd–NADP⁺ reductase (FNR) are redox partners responsible for the conversion between NADP⁺ and NADPH in the plastids of photosynthetic organisms [13], [14]. FNR forms a 1:1 complex with Fd, however, in some proteins of the FNR-family such as phthalate dioxygenase reductase [15] and benzoate dioxygenase reductase [16], Fd-like modules are found in domains fused to the N- or C-terminus of the FNR modules. In our previous study, we prepared a series of cross-linked complexes of maize leaf Fd and FNR by introducing, in each case, a specific disulfide bond between the two proteins, so that the two protein moieties assume various configurations [17]. These Fd–FNR cross-linked complexes showed diverse properties of electron transfer between Fd and FNR. In this study, we solved X-ray crystal structures (Fig. 1) of the two different cross-linked complexes which possessed different unique disulfide bonding sites and distinct electron transfer activity. Unexpectedly, both cross-linked complexes formed oligomers by swapping Fd and FNR moieties across the molecules in crystal. In order to verify whether these oligomeric structures are formed only in crystal or not, we addressed the possibility of the oligomerization of these cross-linked complexes in solution.